Many biological fluids, such as whole blood, plasma, serum, semen, uterine and vaginal fluids, have oxidation-reduction potentials (ORP). Clinically, the ORP of such fluids provides the oxidative status of an animal. More particularly, the ORP of such fluids is related to health, disease and the status of biological processes.
An oxidation-reduction system, or redox system, involves the transfer of electrons from a reductant to an oxidant according to the following equation:oxidant+ne−↔reductant  (1)where ne− equals the number of electrons transferred. At equilibrium, the redox potential (E), or oxidation-reduction potential (ORP), is calculated according to the Nernst-Peters equation:E(ORP)=Eo−RT/nF ln [reductant]/[oxidant]  (2)where R (gas constant), T (temperature in degrees Kelvin) and F (Faraday constant) are constants. Eo is the standard potential of a redox system measured with respect to a hydrogen electrode, which is arbitrarily assigned an Eo of 0 volts, and n is the number of electrons transferred. Therefore, ORP is dependent on the total concentrations of reductants and oxidants, and ORP is an integrated measure of the balance between total oxidants and reductants in a particular system. As such, ORP provides a measure of the overall oxidative status of a body fluid or tissue of a patient.
Oxidative stress is caused by a higher production of reactive oxygen and reactive nitrogen species or a decrease in endogenous protective antioxidative capacity. Oxidative stress has been related to various diseases and aging, it has been found to occur in all types of illnesses, and it has been shown to affect numerous biological processes including conception and pregnancy. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7:161-168 (2004); U.S. Pat. No. 5,290,519 and U.S. Patent Publication No. 2005/0142613; Agarwal et al., Reproductive Biology and Endocrinology, 3:28: 1-21 (2005); Rad et al., J. Caring Sciences, 2(4): 287-294 (2013). Several investigations have shown a close association between the oxidative status of a patient and the patient's outcome. See Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7:161-168 (2004).
Oxidative stress in patients has been evaluated by measuring various individual markers. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7:161-168 (2004); U.S. Pat. No. 5,290,519 and U.S. Patent Publication No. 2005/0142613. However, such measurements are often unreliable and provide conflicting and variable measurements of the oxidative status of a patient. See Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7:161-168 (2004). The measurement of multiple markers which are then used to provide a score or other assessment of the overall oxidative status of a patient has been developed to overcome the problems of using measurements of single markers. See Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7:161-168 (2004). Although such composite approaches are more reliable and sensitive than measurements of a single marker, they are complex and time consuming. Thus, there is a need for a simpler and faster method for reliably measuring the overall oxidative status of a patient.
The oxidation/reduction potential can be measured electrochemically. Electrochemical devices for measuring ORP of blood, blood products, and other biological fluids, typically require large sample volumes (that is, ten to hundreds of milliliters) and long equilibrium periods. Furthermore, the electrochemical devices have large, bulky electrodes that require cleaning between sample measurements. Such electrochemical devices are poorly suited for routine clinical diagnostic testing. It has been suggested to use electrodes that have undergone treatment to prevent biofouling. However, such devices necessarily involve complex manufacturing techniques. Moreover, conventional electrochemical devices have not provided a format that is convenient for use in a clinical setting.
The oxidative and radical characteristics of biological fluids, such as blood plasma and its blood components (such as low density lipoproteins, serum albumin, and amino acids), semen (and its components), uterine and vaginal secretions, can also be determined from photo chemiluminescence, with and without thermo-initiated free radical generation. A photo chemiluminescent system generally includes a free radical generator and a detector that measures chemiluminometric changes in the presence of an antioxidant. More specifically, to measure antioxidant presence, the biological sample (or one of its components) containing an amount of antioxidant is contacted and reacted with a known amount of free radicals. The free radicals remaining after contacting the biological sample are determined chemiluminometrically. Free radicals are measured in a similar chemiluminescent, using a known amount of antioxidants against the endogenous free radicals in the sample. These types of measurements and the detection system are not suitable for rapid, large scale measurements of biological fluid samples in a clinical setting for assessing or monitoring human or animal health.
In recent years, the proportion of men meeting the criteria for normal sperm morphology has dropped substantially. The predictive value of sperm morphology to identify infertile donors from proven donors, ranges from 98.6% to 57.9% (Agarwal, A. et al. (2014). Reprod Biol Endocrinol, 12, 33; Menkveld, R., et al. (2001) Hum Reprod, 16, 1165-1171; Ho, L. M., et al. (2007), J. Androl, 28, 158-163.). Morphology was found to be better at identifying infertile donors than measures of sperm motility, concentration, total sperm count, the hamster egg penetration test (HEPT), or the sperm penetration index (Ombelet, W., et al. (1997). Hum Reprod, 12, 987-993.). More importantly, morphology is strongly related to pregnancy and successful in vitro fertilization (IVF) results with increased odds of implantation and pregnancy, and decreased miscarriages (van den Hoven, L., et al. (2015). Fertil Steril, 103, 53-58; Mortimer, D. and Menkveld, R. (2001). J. Androl, 22, 192-205; Abu Hassan Abu, et al. (2012). Andrologia, 44 Suppl 1, 571-577.). Despite the importance of this parameter, qualitative morphological measures are subject to technical, procedural, and subjective errors more so than other sperm parameter. A common semen sample can produce morphology results that vary as much as 30% (Brazil, C. (2010). Asian Journal of Andrology, 12, 14-20; Auger, J. (2010). Asian Journal of Andrology, 12, 36-46.) Thus, an unbiased, quantitative measure, representative of sperm morphology, would greatly benefit the fertility community.
In some studies, oxidative stress has been associated with infertility in men, and specifically with morphology. (Agarwal, A., et al. (2014). Syst Biol Reprod Med, 60, 206-216; Pons-Rejraji, H., et al. (2009). Gynecol Obstet Fertil, 37, 529-535; Benedetti, S. et al. (2012). Reprod Biomed Online, 25, 300-306; Zorn, B., et al. (2003). Int J Androl, 26, 279-285; Macanovic, B., et al. (2015). Dis Markers, 2015, 436236; Svobodova, M., et al. (2009). Ceska Gynekol, 74, 399-403.) Others studies report a more ambiguous relationship. (Svobodova, M., et al. (2009). Ceska Gynekol, 74, 399-403; Haghighian, H. K., et al. (2015). Fertil Steril; Beresford, M. J., et al. (2010). Clinical Oncology, 22, 46-55.) The difference between these studies are revealed in their methods. Most studies measure a single family of oxidants, i.e. reactive oxygen species (ROS). Others measure the post-hoc damage that accumulates under oxidative stress, i.e. lipid peroxidation. Lastly, the changes in antioxidant levels or activity have been used, i.e. total antioxidant capacity (TAC) or superoxide dismutase (SOD). With the numerous ways in which “oxidative stress” is defined by single measures, it is not unreasonable to question the role of oxidative stress in male fertility. Oxidative stress is a state in which the activity of the oxidants exceeds the capabilities of the antioxidants to quench them. Thus, there is a need for a simple measure that compares all oxidant activity to all antioxidant activity as a predictor of sperm morphology and infertility. The present invention addresses this need and provides other, related benefits as well.